Variation in insulative feather structure in songbirds replacing each other along a tropical elevation gradient

Abstract High‐elevation organisms are expected to evolve physiological adaptations to cope with harsh environmental conditions. Yet, evidence for such adaptive differences, especially compared to closely related lowland taxa occurring along the same elevational gradient, is rare. Revisiting an anecdotal natural history observation by O. Bangs from 1899 and based on new measurements of museum specimens, we confirmed that the high‐elevation hermit wood wren (Henicorhina anachoreta) from the Sierra Nevada de Santa Marta, Colombia, has longer, more insulative feathers on the chest and back, than its lower‐elevation counterpart the grey‐breasted wood wren (H. leucophrys). However, we did not find evidence for the same specializations in subspecies of H. leucophrys that live at high elevations on other elevational gradients in the Colombian Andes, although similar adaptive solutions have arisen in separate mountain systems like the Himalayas. Adaptations in plumage may be associated with the recurrence of elevational species replacements throughout the tropics.


| INTRODUC TI ON
How do tropical organisms living at high elevations cope with the demands imposed by such harsh environments? Answering this question is crucial to understand the elevational range limits of species and to forecast their responses to climatic change (Jankowski et al., 2013;Linck et al., 2021). An example of the challenges posed by living at high elevations is the reduced partial pressure of oxygen, which has prompted numerous physiological adaptations in vertebrates (Storz, 2021). Although hematological adaptations to high elevation in birds have been well characterized (e.g., Barve et al., 2016), how birds cope with low temperatures at high elevations has been much less studied and the mechanisms conferring tolerance to cold conditions in tropical species are not well understood (Londoño et al., 2017).
A likely axis along which bird species from tropical mountains may adapt to cold temperatures in the highlands is via increased insulation provided by their plumage (Gamero et al., 2015). For example, in the original taxonomic description of the hermit wood wren (Troglodytidae, Henicorhina anachoreta) from the Sierra Nevada de Santa Marta, northern Colombia, Outram Bangs outlined an intriguing adaptive hypothesis. When comparing the high-elevation taxon anachoreta to parapatric populations from lower elevations, now referable to his namesake grey-breasted wood wren (H. leucophrys bangsi, see Caro et al., 2013;Cadena et al., 2016), Bangs (1899) subjectively noted that plumage was "everywhere, much longer and denser, but especially noticeably so on the head and neck, the bird evidently being fitted to withstand cold weather." The possibility that birds from higher elevations in Santa Marta may have plumage adapted to cold temperatures is tantalizing given their smaller body size compared to birds from lower elevations (Cadena et al., 2016), which is contrary to the prediction that endotherms should increase relative body surface area in cold environments to reduce heat loss through thermal conductance to the environment (i.e., Bergmann's rule; Freeman, 2017).
The role of plumage providing insulation has long been recognized in studies of climatic adaptation in birds (Scholander, 1955) and several examples point to variation in feather structure associated with living in cold environments (de Zwaan et al., 2017;Koskenpato et al., 2016;Pap et al., 2020). In particular, a recent study documented a widespread increase in the downy section of contour feathers and in contour feather length with elevation in passerine birds from the Himalayas, revealing patterns of convergent evolution resulting in more insulative feathers and plumage at higher elevations across several passerine families (Barve et al., 2021). The proportion of down increases insulative capacity in each feather, whereas longer feathers give rise to a deeper, more insulative overall plumage (Pap et al., 2017). Barve et al. (2021) showed that Himalayan taxa may show one or both traits along elevational gradients. However, not all species examined showed variation in feather structure across the elevational gradient (Barve et al., 2021), suggesting that plumage modifications may or may not arise as a result of the effect of factors such as a species' metabolic flexibility (Londoño et al., 2015), gene flow across the elevational gradient swamping adaptive variation (Bridle et al., 2009), or plasticity or lack thereof in the trait itself (Price et al., 2003).
Elevational replacement of closely related bird species is a pervasive pattern in tropical mountains across the world (reviewed by Cadena & Céspedes, 2020). Several ecological hypotheses have been proposed to account for such patterns of replacement, with a common conjecture being that the physiological stress of cold temperatures, at least in part, sets the upper elevational limit of the low-elevation species, where it is replaced by its high-elevation counterpart (Barve & Dhondt, 2017;Freeman et al., 2019;Terborgh, 1977). However, case studies where adaptation to cold temperature is demonstrated in the highland species in an elevationally replacing species pair are rare (Jankowski et al., 2013).
The Sierra Nevada de Santa Marta is widely regarded as the tallest coastal mountain on Earth, rising from sea level in the hot Caribbean lowlands to snow-covered peaks corresponding to the highest elevations in Colombia (Figure 1a). Wood wren species in the genus Henicorhina replace each other with elevation in Santa Marta (Caro et al., 2013;Todd & Carriker, 1922), with H. leucophrys occurring in warm forest environments typically below 2000 m and H. anachoreta in colder cloud forests and elfin forests reaching more than 3000 m (see Cleef et al. (1984) for details on climatic and eco-  Paynter et al. (1997)). (b) Data on mean minimum temperature in Colombia between 1971 and 2000 indicate the Sierra Nevada de Santa Marta and Andes have similar patterns of temperature variation with elevation. Temperature data were downloaded from WorldClim 2.1 (Fick & Hijmans, 2017). The map was generated using the R packages ggplot2 (Wickham, 2016), sf (Pebesma, 2018), maps (Brownrigg, 2013), and elevatr (Hollister et al., 2017) whether the pattern described subjectively by Bangs (1899) is verified by quantitative analyses focused on variables reflecting the insulative properties of feathers, that is, whether the higher-elevation species H. anachoreta has more insulative plumage than the lowerelevation species H. leucophrys.
On elevational gradients in the Colombian Andes exhibiting similar temperature variation to that existing in the Sierra Nevada de Santa Marta (Figure 1b

| Statistical analyses
For 34 specimens (10 each of H. anachoreta, H. l. bangsi, and H. l, leucophrys, and 2 each of H. l. brunneiceps and H. l. manastarae; i.e., 33% of all specimens used in the study), we ensured repeatability of all feather structure measurements described above by measuring the same variables on two adjacent feathers and using the R package Caper to estimate the interclass correlation coefficient (Orme et al., 2013). Both feather measurements had high repeatability (interclass correlation coefficient) within an individual (proportion of downy section: chest = 0.87, back = 0.83; and total feather length: Because H. anachoreta is smaller than H. leucophrys (Cadena et al., 2016), we controlled for body size in feather length measurements by dividing the total feather length by the median body mass of the species measured in an earlier study (Caro et al., 2013).
While testing for differences between high-and low-elevation specimens of H. leucophrys, we corrected for differences in body size by dividing the total feather length by the tarsus length of the specimen. Therefore, all analyses were done using measures of relative feather length. To reduce redundancy in our analyses, we first confirmed that there was little correlation between proportion of downy section and feather length for the chest (r = .14, p = .15) or back feathers (r = .03, p = .33). Next, we used these two variables to test for variation in feather structure with elevation in the two elevationally replacing species from the Sierra Nevada de Santa Marta using linear multiple regressions. In each multiple regression, the feather structure variable (either proportion down or feather length) was the dependent variable and an interaction of the species ID and elevation was the independent variable. Through this analysis, we wanted to explore whether one or both species showed within-species variation in feather structure with elevation (as a likely response to temperature) and whether the slopes of the relationships between elevation and feather structure varied between species.
We also used multiple regressions to test for differences in proportion of downy section and feather length in the lowland H.l.
bangsi and highland specimens of H. leucophrys collected from other regions of its range. In all cases, we did the analyses separately for chest and back feathers. All analyses were done in the R Statistical Programming Language 4.0.5 (R Core Team, 2021).

| RE SULTS
Patterns of variation in feathers within and between species along the elevational gradient in the Sierra Nevada de Santa Marta were consistent with adaptive plumage scenarios given the occurrence of specimens of H. leucophrys at elevations lower than those of H. anachoreta (Table 1; Figure 2a). On the chest feathers, the proportion of downy section did not vary with elevation in either species, but the highland H. anachoreta had overall longer feathers relative to body size (p = .03, Table 1, Figure 2c). On the back, as on the chest, the proportion of the downy section in feathers did not change with elevation. However, H. anachoreta had significantly longer back feathers (p < .001, Table 1, Figure 2e) than H. leucophrys, the low-elevation species. Relative feather length increased with elevation significantly more steeply in H. anachoreta than in H. leucophrys for back feathers (significant interaction term between elevation and species, p = .02, Table 1, Figure 2e). On both TA B L E 1 Result of linear multiple-regression models testing the role of an interaction between elevation and species in explaining the variation in feather structure in wood wrens from the Sierra Nevada de Santa Marta. Significant differences (p < .05) are highlighted.

| DISCUSS ION
Our quantitative analyses of feather structure based on museum specimens allowed us to confirm Bangs' (1899) anecdotal observation that high-elevation Henicorhina wood wrens from the Sierra Nevada de Santa Marta may have longer, more insulative plumage than their lower-elevation counterparts to cope with cooler temperatures. Himalayan birds increase the insulation provided by their plumage via increasing the proportion of down in their feathers or by increasing the length of their feathers, which thus overlap more extensively with neighboring feathers to produce a denser, more insulative plumage (Barve et al., 2021).
We found that Henicorhina wood wrens in Santa Marta seemingly use this second strategy, in agreement with Bangs' (1899) hypothesis.
We did not find a significant increase in the proportion of down in either the chest or back contour feathers, but did see relatively Elevational species replacement is a ubiquitous global pattern in many groups of organisms, particularly in tropical mountains (Cadena & Céspedes, 2020). Although lower-elevation species are often competitively superior to high-elevation taxa (Jankowski et al., 2010; but see Freeman, 2020), the upper elevational range limits of species from lower elevations may be set by physiological constraints, restricting their elevational ranges, and allowing their coexistence in the same mountain slopes with species better adapted to the harsh conditions of high elevations. Nevertheless, evidence for differences in phenotypic characters that may directly confer a physiological advantage to high-elevation species is rare (Gaffney, 2017). Multiple factors determine the elevational distribution of species (Sexton et al., 2009), but temperature is an important factor, especially for small endotherms like birds (Elsen et al., 2016;Tingley et al., 2009). Our results show that in the elevationally replacing Henicorhina wood wrens of the Sierra Nevada de Santa Marta, the highland H. anachoreta indeed has more insulative plumage than the  (Caro et al., 2013). Assuming that such bill and body dimensions are strongly heritable, the fact that feather structure varies more gradually indicates that this trait may be more plastic, dependent more on the elevation individuals live at, rather than on species-specific genetic factors.
Populations in the H. leucophrys complex occurring at high elevations in the Sierra Nevada de Santa Marta (Caro et al., 2013) and the Andes of Ecuador (Dingle et al., 2008(Dingle et al., , 2010  In conclusion, elevationally replacing wood wrens in the Sierra Nevada de Santa Marta (but not in the Andes) showed variation in feather structure with elevation similar to that shown by phylogenetically distant Himalayan birds. This suggests that feather modifications that increase their thermo-insulative potential may be a widespread adaptation in the world's montane birds. Expanding our study to a larger set of South American birds would allow to further confirm this generality. Our work underscores the immense value of museum collections in the comparative research of morphological trait evolution. Natural history collections provide critical access to biological materials where newly developed techniques can be used to comprehensively test classical eco-evolutionary hypotheses.

ACK N OWLED G M ENTS
We would like to thank Terry Chesser, Carla Dove, and Chris Milensky for help with logistics at the Smithsonian National Museum of Natural History and the Peter Buck Fellowship Program for support for SB. We would also like to thank the Cadena lab and three anonymous reviewers for constructive feedback on the manuscript.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are made available as supplementary material.